Degeneration of tissue, inflammation and wound healing induced by non-immunological and/or by immunological processes lead to the formation of a variety of endogenous, biologically active substances (mediators and hormones). Mostly, their nature and mode of action have not been characterized in detail so far. They regulate the complex individual steps of activation of inflammation and tissue repair processes and thus participate in homeostasis and turnover of tissues. Accumulation of leukocytes by chemoattraction, altered hemodynamics, smooth muscle contraction, fever, angiogenesis, leukocytosis, leukopenia and/or leftward shift reactions, but also shock symptoms, are examples of classical phenomena in physiopathology which can be mediator-induced and which are operative in and/or accompany systemic and local defense and repair mechanisms in inflammation, wound healing, tumor growth and reproductive cycles. These substances are produced either as humoral mediators by limited and regulated proteolysis of serum and plasma proteins; or they are released as cellular mediators from cells and tissues by cell lysis or active (autocrine, paracrine or endocrine) secretion. They form part of the body's defense system, the systemic and local activation of which they regulate. Thus, they contribute to removal and detoxification of destroyed endogenous substances and/or of invaded exogenous foreign bodies. In addition, by regulation of the cell-division and tissue growth processes in wound healing they participate in restoration of physiological structures and functions of the organism. Like the classical hormones of endocrine glands, inflammatory mediators are trace substances that are present in situ in very minute concentrations in tissue or blood. So far, however, once isolated in a biologically specific active form, they also can be considered and may be useful as ideal, valuable (natural) drugs of high specificity of action and lack of undesired side-effects (intrinsic to synthetic drugs), since they have been formed by, and underwent natural selection in evolution.
By activation of the kinin system, the coagulation system, the complement system and also of other blood protein and cell factors, a variety of humoral mediators may be produced concurrently or sequentially which are responsible for the numerous apparent biological activities of the activated serum. The leukocytosis reaction (reactive mobilization and recruitment of leukocytes from the bone marrow into the blood with reactive increase of blood cell number over normal level) is also such a mediator-triggered reaction. The recruitment of leukocytes from the bone marrow is a common feature of acute inflammatory processes, for example in bacterial infections or myocardial infarction. The leukocytosis reaction constitutes part of feed-back mechanism in regulatory loops of the body's defense system to maintain the structural and functional readiness of the organism for tissue repair. Hence impaired function in leukocyte mobilization mechanisms, e.g. in virus infections or genetic defects (congenital genetic agranulocytosis) may have fatal prognosis.
Two negative feedback mechanisms have been postulated for homeostasis (maintainance of the normal range) of constant blood leukocyte concentration. One shall be responsible for production of cells in the bone marrow and the other for their release into the blood; c.f. D. R. Boggs, Ann.Rev., Physiol., vol. 28 (1966) p. 39-56. Humoral mediators are thought to play a part in these regulatory loops; cf. V. Menkin, Biochemical Mechanisms in Inflammation, 2nd edition, Charles C. Thomas, Springfield, Ill., 1956. In this work, Menkin also showed for the first time that soluble mediators are likewise involved in mechanisms, which increase the blood leukocyte concentration above the normal level range (leukocytosis reaction). He isolated a crystallizable protein-like material from inflammatory exudates which induced a leukocytosis reaction in vivo, but which was not identified and characterized in detail.
These early experiments provided a valuable basic knowledge on formation, nature and possible mode of action of inflammatory mediators. However, so far, on the one hand it was impossible to rule out possible contaminations of the material with bacterial endotoxins which may mimic non-specifically and indirectly through mediator formation the activities ascribed to the protein preparations [cf. D. R. Boggs, op. cit. (1966)]. On the other hand, it could not be excluded that contaminations with other trace proteins caused artefact reactions, since present methods for selective purification of complex mixtures and analysis of proteins for molecular homogeneity were not available at that time.
Many different factors can trigger leukocytosis reactions in the intact organism. Amongst them to mention are infections, immune reactions, mechanical tissue damage, drugs (e.g. cortisone), mental stress, or even the consumption of extensive meals. However, leukocytosis reaction may be induced when leukocytes are recruited from marginated (secondary) storage pools in tissues or from their production site, the bone marrow, which also constitutes a (primary) storage pool for leukocyte recruitment; cf. H. E. Whipple and M. I. Spitzer, Ann. N.Y. Acad. Sci. 113 (1964) pp. 511-1092. Hence, increase of blood leukocyte concentration (leukocytosis) is not simply identical to mobilization of leukocytes from bone marrow.
Whether or not such triggers act directly or indirectly (by formation of specific mediators) on the bone marrow and its leukocyte storage pools remained a matter of debate, and has to be established from case to case. A main indirect trigger in infections are endotoxins (lipopolysaccharides) which exert many biological effects by activating humoral (e.g. the serum complement system) and cellular defense system (e.g. B-lymphocytes, monocytes etc.). Thus, their indirect effects on hematopoesis earlier served as to assay them; cf. O. Luderitz, Angew. Chem. 82, (1970), pp. 708-722. Hence, a main problem intrinsic to research in basic mechanisms of leukocyte mobilization from the bone marrow into blood circulation, thus, was the development of reliable in-vitro test systems, the results of which can be correlated to in-vivo leukocytosis reaction test systems. A. S. Gordon et al. [Ann. N.J. Acad. Sci., vol. 113 (1964) p. 766-789] developed such a laborious in-vitro test system with which they obtained a leukocytosis-inducing plasma factor. Its nature has not been clarified in greater detail, and its function rather must be considered as part of the feed-back mechanisms that might correct reduced blood leukocyte concentrations (leukopenia) to normal levels.
K. Rother [Eur. J. Immunol., vol.2 (1972), pp. 550-558] developed another reliable, but less laborious in-vitro test system for the detection of leukocyte mobilization from the bone marrow. Rother was the first to put forward the argument that a humoral factor from the third component of the complement system plays a part in the leukocytosis reaction. This finding led to protein preparations, the nature, molecular homogeneity and/or biological specificity of which, however, were not further characterized in detail. In addition, their preparation methods were not considered for obtaining physical quantities for use in practice of a mediator of the leukocytosis reaction. Recent experience with inflammatory mediators shows [cf. J. H. Wissler, Eur. J. Immunol., vol. 2 (1972), pp. 73-96] that these are trace substances active in the nanomolar concentration range. Hence, for their preparation in molecularly homogenous form and in physical quantities, purification techniques used in multi-step sequences are necessary which can easily handle large volumes of the starting material.
For example, one of these protein preparations was obtained by activation of the serum complement system in small volumes of serum of small animals followed by a sequence of steps of fractional salting-out precipitation with ammonium sulfate, an electrophoresis and a molecular sieve chromatography; cf. K. Rother op. cit. This protein preparation induces the release of leukocytes from an isolated rat femur in vitro and a leukocytosis reaction in vivo. The in vivo leukocytosis reaction seems to be complex. It is biphasic and can be inhibited only in part by an antibody serum preparation to the complement component C3; [cf. K. Rother et al., Z. Immun.-Forsch.-Immunobiology, vol 155 (1978), p. 55].
A similar protein preparation (molecular weight 10,000 to 12,000 dalton) has been prepared by B. Ghebrehiwet and J. H. Muller-Eberhard, J. Immunol., vol. 123 (1979), pp. 616-621, by digestion with trypsin of a preparation of complement component C3. This preparation was purified by electrophoresis or alternatively by means of an ion exchange step and electrophoresis. The protein preparation contains no tyrosine as amino acid structural component; cf. B. Ghebrehiwet and J. H. Muller-Eberhard, op.cit. (1979). It mobilizes leukocytes in vitro and induces a leukocytosis reaction in rabbits in vivo with a specific activity of 10 .mu.g/kg (1 mol/kg). In addition, it was found that this preparation acts non-specifically and has more than one biological activity: It also increases capillary permeability in situ and exerts a local phlogistic action (emigration of leukocytes into the tissue); cf. B. Ghebrehiwet and J. H. Muller-Eberhard, op.cit. (1979).
A different biologically active factor inducing a very late monocytosis reaction (after 24-48 hours) without affecting granulocyte levels in mice has been detected in murine serum during the early phase of an inflammatory reaction, having a molecular weight of 18,000 to 24,000 dalton; [cf. D. van Waarde et al., Blood, vol 50 (1977), pp. 727-742]. Whether or not this factor is derived from serum proteins or is of cellular origin has not been clarified and its purification and further characterization waits to be established.
Measurement of leukocyte mobilization from the bone marrow and leukocyte recruitment into blood on the one hand is carried out biologically, either in vitro according to the test system of K. Rother (rat femur) [Eur. J. Immunol., vol.2 (1972), pp. 550-558]; or in vivo by time-dependent periodic counting and differentiation of the various types of leukocytes in the blood after administration of a solution of the substance under investigation. On the other hand, the leukorecruitin can be measured physico-chemically by the various usual immunochemical methods (e.g. immunodiffusion immunoelectrophoresis, or radioimmunoassay) using an anti-leukorecruitin immunoglobulin preparation. It can be prepared from the molecularly homogeneous, purified leukorecruitin in accordance with the invention. With these test systems leukocyte-recruiting activity can be discovered in serum or plasma after their activation by contact reactions with many high molecular, exogenous substances (such as immune complexes, endotoxins, or snake venoms), microorganisms (e.g. Pseudomonas), and proteolytic enzymes. Similar findings have been obtained by K. Rother, op. cit (1972) and B. Ghebrehiwet and J. H. Muller-Eberhard, op. cit (1979). However, in certain cases long incubation periods (e.g. 5 days) are necessary.
It is therefore a primary object of this invention to provide a natural leukocytosis-inducing (leukocyte-recruiting and mobilizing) protein from mammalian serum.
It is another object of this invention to provide a natural leukocytosis-inducing protein from mammalian serum in molecularly homogeneous crystallizable form.
It is another object of this invention to provide a natural leukocytosis-inducing protein from mammalian serum, which represents a biologically specific, active and natural mediator of the leukocytosis reaction.
It is another object of this invention to provide a natural leukocytosis-inducing protein from mammalian serum, which is suitable for specifically influencing the defense state of mammalian (e.g. human) organism.
It is still another object of this invention to provide a process for producing and obtaining a natural leukocytosis-inducing protein from mammalian serum plasma or blood.
It is still another object of this invention to provide a process for producing and obtaining a natural leukocytosis-inducing protein from mammalian serum plasma or blood in an economical, biotechnically useful and relatively simple manner.
It is still another object of this invention to provide a process for producing and obtaining a natural leukocytosis-inducing protein from mammalian serum plasma or blood in a highly purified, molecularly homogeneous, crystallizable form and in physical quantities for practical use.
These and other objects and advantages of the present invention will be evident from the following description of the invention.